Polyethylene glycol precipitation is an efficient method to obtain extracellular vesicle-depleted fetal bovine serum.

Autor: Wang P; Experimental Rheumatology, Radboud University Medical Center, Nijmegen, Netherlands., Arntz OJ; Experimental Rheumatology, Radboud University Medical Center, Nijmegen, Netherlands., Husch JFA; Department of Dentistry Regenerative Biomaterials, Radboud University Medical Center, Nijmegen, Netherlands., Kraan P M V; Experimental Rheumatology, Radboud University Medical Center, Nijmegen, Netherlands., Beucken JJJPVD; Department of Dentistry Regenerative Biomaterials, Radboud University Medical Center, Nijmegen, Netherlands., van de Loo FAJ; Experimental Rheumatology, Radboud University Medical Center, Nijmegen, Netherlands.
Jazyk: angličtina
Zdroj: PloS one [PLoS One] 2023 Dec 05; Vol. 18 (12), pp. e0295076. Date of Electronic Publication: 2023 Dec 05 (Print Publication: 2023).
DOI: 10.1371/journal.pone.0295076
Abstrakt: Mesenchymal stromal/stem cell derived-extracellular vesicles (MSC-EVs) have gained interest as drug delivery nanoparticles, having immunoregulatory and potentiating tissue repair property. To maintain growth of MSCs and obtain pure MSC-derived EVs, the culture media should contain fetal bovine serum (FBS) devoid of EVs, as the presence of FBS EVs confounds the properties of MSC-EVs. Therefore, we tested three methods: 18h ultracentrifugation (UC) and ultrafiltration (UF), which are common FBS EV depletion methods in current MSC-EV research, and polyethylene glycol (PEG) precipitation to obtain three EV depleted FBS (EVdFBS) batches, and compared them to FBS and commercial (Com) EVdFBS on human adipose stem cell (hADSC) growth, differentiation, enrichment of EVs in hADSC supernatant and their biological function on collagen metabolism. Our comparative study showed UC and UF vary in terms of depletion efficiency and do not completely deplete EVs and affects the growth-promoting quality of FBS. Specifically, FBS EV depletion was comparable between PEG (95.6%) and UF (96.6%) but less by UC (82%), as compared to FBS. FBS protein loss was markedly different among PEG (47%), UF (87%), and UC (51%), implying the ratio of EV depletion over protein loss was PEG (2.03), UF (1.11), and UC (1.61). A significant decrease of TGFβ/Smad signaling, involving in MSC growth and physiology, was observed by UF. After 96 hours of exposure to 5% FBS or 5% four different EVdFBS cell growth media, the osteogenesis ability of hADSCs was not impaired but slightly lower mRNA expression level of Col2a observed in EVdFBS media during chondrogenesis. In consistent with low confluency of hADSCs observed by optical microscope, cell proliferation in response to 5% UF EVdFBS media was inhibited significantly. Importantly, more and purer ADSCs EVs were obtained from ADSCs cultured in 5% PEG EVdFBS media, and they retained bioactive as they upregulated the expression of Col1a1, TIMP1 of human knee synovial fibroblast. Taken together, this study showed that PEG precipitation is the most efficient method to obtain EV depleted FBS for growth of MSCs, and to obtain MSC EVs with minimal FBS EV contamination.
Competing Interests: NO
(Copyright: © 2023 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
Databáze: MEDLINE
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